The radio labelling of antibodies, proteins or peptides to produce efficient imaging agents remains a very important goal in the field of molecular imaging which is presently hindered by the lack of suitable compounds.
The natural ability of native molecules to target their antigens or receptors could lead to highly specific imaging or therapeutic molecules due to their innate affinity and specificity. Imaging agents could, in principle, be obtained from such peptides or proteins via their labelling with an element possessing a property that renders these labelled molecules detectable with an imaging system.
Among the various methods developed, radio labelling with 99mTc has been long recognized as a very attractive approach. 99mTc is a metastable nuclear isotope of technetium-99, which has almost ideal properties for in vivo diagnostic imaging. It has a half life (about 6 h) which is long enough to allow the preparation of an imaging agent and for the diagnostic studies to be performed, but is short enough to permit the administration of millicurie amounts without exposing the patient to a significant dose of irradiation.
The monochromatic 140 Key photons and its 89% abundance are ideal for imaging studies with gamma cameras or SPECT systems. In addition, 99mTc can be easily obtained from 99Mo/99mTc generator taking advantage of the transient equilibrium between the parent radionuclide 99Mo and the 99mTc radionuclide. The separation of 99Mo and 99mTc is easily achieved from the generator by elution of pertechnetate TcO4− with saline solutions.
The Tc cores can then be prepared by reduction of the eluted pertechnetate. Several oxidation states of Tc can be generated and used. One of the most studied core corresponds to diatomic Tc(V) core [TcO]3+ and more recently a number of studies reported the production and use of the Tc(I) triscarbonyl [Tc(CO)3]+ to label peptides and proteins.
Rhenium has similarly advantageous properties which make it a suitable element for radiolabelling and in vivo/in vitro imaging and therapeutic uses. In addition Re and Tc have very similar chemistry and as Re is less expensive to produce and certain of its isotopes have a longer half-life than Tc, this could make Rc a better radionuclide for some uses.
Several strategies have already been proposed to radiolabel peptides and proteins with technetium or rhenium:                A first method corresponds to the so-called direct method, which involves the reduction of disulfide bridges present in the molecule of interest and the chelation of the Tc core to the resulting sulfudryl groups (1). While this first method appears simple and easy to perform, it has a number of limitations (2). The first one concerns the impact of chelation on the structure of the protein, normally disulfide bridges play an important role in forming and maintaining the correct folding/structure of a protein and therefore its binding capacity to the targeted receptor. By disrupting the disulfide bridges the resulting labelled protein can lack affinity for its target. Furthermore, the chelated Tc core may interact with or be positioned in the binding region of the protein of interest resulting in the partial or total loss of its biological activity.        A second, indirect method has been proposed to label protein and peptides with Tc cores using a bifunctional chelating agent (BFCA). This method is based on the chemical grafting of strong Tc core chelators onto the surface of the protein by a chemoselective reaction that most often involves Lysine residues. One of the most widely used chelator is the HYNIC group (hydrazinonicotinamide) (3,4). While the presence of such a strong chelator increases the labelling yield and the resulting stability of the Tc complexes formed, the major drawback of this method is that it offers no control on the location of the BFCA on the surface of the protein, which thus can lead to a pool of heterogenous labelled molecules a subset of which may have reduced binding capacity and in any event make quantitative and repeatable studies difficult.        A third approach, called preformed chelate has been proposed to label antibodies (5, 6). However, this approach suffers from the same limitations as the direct and the indirect approaches regarding the absence of control over the location of the preformed Tc-Chelate complex that reacts with any Lysine residues present.        
Site specific labelling of proteins with Tc or Re using peptide tag sequences has been proposed as an alternative to the methods described above. This approach consists of adding at a N-terminal or C-terminal position of the protein of interest, a peptide sequence able to chelate a Tc/Re core. Several peptide sequences have been described as [TcO]3+ chelators (7). These peptides form square pyramidal oxo-technetium complexes with tetradentate chelators. The majority of the reported natural amino acid containing peptides possess a Cysteine residue that participates in the chelation via its thiolate group.
Examples of this type of peptide tag are the Gly-Gly-Cys (SEQ ID NO: 28) and the Lys-Gly-Cys (SEQ ID NO: 29) sequences with a N3S chelating motif and the Cys-Gly-Cys (SEQ ID NO: 30) with a N2S2 motif (8, 9). It is however known in the art that the presence of an unpaired Cysteine may interfere in the folding processes of a protein and therefore reduce the yield of production of the modified protein or alter its final conformation. An unpaired Cysteine may also lead to alteration of the targeting molecule during storage.
[TcO]3+ chelating-peptide sequences containing proteinogenic amino acids but without Cysteine residues would therefore appear to be good candidate molecules. However, such peptide sequences are extremely uncommon and in the art only peptide sequences containing Glycine and Alanine residues have been shown to be able to chelate the [TcO]3+ core in a N4 motif (10, 11). These peptides showed interconverting [TcO]3+ syn and anti isomers and no stability data has been reported for these complexes prior to in vivo testing. Stability is an essential property of a radio-labelled reagent as dissociated [TcO]3+ complexes will result in a greater non-specific background signal leading to a lower signal to noise ratio.
In addition, it was recently demonstrated that the [AGGG]TcO complex (SEQ ID NO: 31) was completely unstable against 30 equivalents of Cysteine (12).
Recently dipeptide sequences have been proposed as TcO/ReO chelating sequence (13). These dipeptides have the major disadvantage that they comprise only three Tc/Re chelating functional groups, which would be expected to impact upon the stability of the complex, thereby reducing their usefulness for in vivo applications.